1. Field of the Invention
The present invention relates to an optical signal detection method for detecting a specific substance in a sample by detecting an optical signal output from a label. The optical signal detection method includes a fluorescence detection method for detecting a specific substance in a sample by detecting fluorescence. Further, the present invention relates to an optical signal detection apparatus, a sample cell for detecting an optical signal, and a kit for detecting an optical signal.
2. Description of the Related Art
Conventionally, in the field of bio-measurement or the like, a fluorescence detection method is widely adopted as a highly accurate and easy measurement method. In the fluorescence detection method, a sample that is supposed to include a detection target substance that outputs fluorescence by being excited by irradiation with light having a specific wavelength is irradiated with the excitation light having the specific wavelength. At this time, the fluorescence is detected to confirm the presence of the detection target substance. Further, when the detection target substance per se is not a phosphor (fluorescent substance), a substance that has been labeled with a fluorescent dye and that specifically binds to the detection target substance is placed in contact with the sample. Then, fluorescence from the fluorescent dye is detected in a manner similar to the aforementioned method, thereby confirming the presence of the bond between the detection target substance and the substance that specifically binds to the detection target substance. In other words, presence of the detection target substance is confirmed, and this method is widely adopted.
Further, in the fluorescence detection method as described above, a method utilizing an electronic field enhancement effect by plasmon resonance to improve the sensitivity of detection is proposed in U.S. Pat. No. 6,194,223 (Patent Literature 1) or the like. In the method disclosed in Patent Literature 1, a sensor chip including a metal layer (metal film, foil or coating) deposited in a predetermined area of a transparent support body is provided. Further, excitation light is caused to enter the interface between the support body and the metal layer at a predetermined angle greater than or equal to a total reflection angle. The excitation light is caused to enter the interface from a surface of the support body, the surface being opposite to the metal-layer-formed surface of the support body. Accordingly, surface plasmons are generated in the metal layer by irradiation with the excitation light. Consequently, fluorescence is enhanced by the electric field enhancement action by the surface plasmons, thereby improving the S/N (signal to noise) ratio.
However, in a surface-plasmon-enhanced fluorescence detection apparatus, when the fluorescent dye in the sample and the metal layer are too close to each other, a problem as described in F. Yu et al., “Surface Plasmon Fluorescence Immunoassay of Free Prostate-Specific Antigen in Human Plasma at the Femtomolar Level”, Analytical Chemistry, Vol. 76, Issue 22, pp. 6765-1770, 2004 (Non-Patent Literature 1) may arise. Specifically, energy excited in the fluorescent dye transfers to the metal layer before fluorescence is produced by the energy excited in the fluorescent dye. Therefore, fluorescence may not be produced (so-called metal-quenching may occur).
Therefore, Non-Patent Literature 1 proposes a method of forming a carboxylmethyl dextran (CMD) coating on the metal layer to maintain a certain distance between the fluorescent dye and the metal layer.
However, in the method disclosed in Non-Patent Literature 1, when the CMD coating is formed on the metal layer, it is necessary to apply the CMD coating after an SAM (self-assembled monolayer) coating is applied to the metal layer. Therefore, longer time and additional steps are required to prevent metal-quenching. Further, it is difficult to control the position of the CMD coating to which the fluorescence-labeled substance is attached and to strictly control the distance between the fluorescent label and the metal layer. When the distance between the fluorescent label and the metal layer is not controlled as intended, the intensity of the fluorescent signal is greatly influenced, thereby deteriorating the reliability of the signal.
The problem of metal quenching and other problems arising therefrom are not limited to the case of using the fluorescent label. Similar problems also occur when a photo-reactable substance having a certain photo-reactable characteristic to light is used as a label.